Because unequal crossing-over is thought to play an essential role in the evolution of repeated gene families, we have devised means to study the genetic recombination behavior of the oocyte-type 5S RNA gene family (5S DNA) from Xenopus frogs. By placing segments of 5S DNA between selectable markers in bacteriophage lambda cloning vectors, we can select for recombinants in which the crossover event must have occurred entirely within 5S DNA. We are specifically testing the hypothesis that internal 15 bp repeats in the 5S DNA spacer function to create a recombinational hotspot. These experiments are being carried our first in bacteria, where the properties of the bacterial (Rec) and bacteriophage (Red) recombination systems are being investigated separately. The recombination behavior of 5S DNA in a homologous Xenopus recombination system will be studied by injecting the 5S DNA-containing lambda DNAs into living oocyte nuclei, then assaying for and characterizing recombinants by selection and amplification in bacteria. The molecular details of the crossover events will be studied by nucleotide sequence analysis of selected recombinants. Another goal is to discover unique features of the chromatin structure of the oocyte-specific 5S DNA genes when they are being transcribed. The nucleosome structure of 5S DNA in oocytes will be examined, as will the susceptibility of specific sites in the 5S DNA repeating unit to restriction endonucleases, and the results compared with inactive 5S chromatin structure in somatic cells. Correlation will be made with sites in 5S DNA known or suspected to be involved in transcription initiation and control. Finally, two satellite DNAs, with repeating units of 800 bp and 420 bp, have been isolated from the X. laevis genome; and their recombination behavior and chromatin structure will be compared with those of 5S DNA.